Amino terminal acylation is an important co-translational modification of proteins in prokaryotic and eukaryotic cells. Although formyl, pyruvoyl, .alpha.-ketobutyryl, glycosyl, glucuronyl, .alpha.-aminoacyl, p-glutamyl, myristoyl, and acetyl are well-known N.sup..alpha. -acylating groups, it is clear that acetylation is the most common chemical modification of the .alpha.-NH.sub.2 group of eukaryotic proteins (Tsunasawa, S., et al., Methods Enzymol 106:165-170 (1984); Driessen, H. P. C., et al., CRC Crit. Rev. Biochem 18:281-325 (1985)).
N.sup..alpha. -acetylation plays a role in normal eukaryotic translation and processing (Wold, F., Trends Biochem. Sci. 9: 256-257, (1984)), and protects against proteolytic degradation (Jornvall, H., J. Theor. Biol. 55:1-12 (1975); Rubenstein, P., et al., J. Biol. Chem. 254:11142-11147 (1979)). Further, the rate of protein turnover mediated by the ubiquitin-dependent degradation system depends on the presence of a free .alpha.-NH.sub.2 group (Hershko, A., et al., Proc. Natl. Acad. Sci. U.S.A. 81:7021-7025 (1984); Bachmair, A., et al., Science 234:179-186 (1986)), and this dependence indicates that N.sup..alpha. -acetylation may play a crucial role in impeding protein turnover.
After the discovery that an acetyl moiety was the N-terminal blocking group of tobacco mosaic virus coat protein (Narita, K., et al., Biochim. Biophys. Acta. 28:184-191 (1958)), and .alpha.-melanocyte-stimulating peptide (Harris, J. I., et al.. Biochem J. 71:451-459 (1959)), a large number of proteins from various organisms have been shown to possess acetylated N-terminal residues (Brown, J. L., et al., J. Biol. Chem. 251:1009-1014 (1976); Brown, J. L., et al., J. Biol. Chem. 254:1447-1449 (1979)). For example, mouse L-cells and Ehrlich ascites cells have about 80% of their intracellular soluble proteins N.sup..alpha. -acetylated (Brown, J. L., et al., J. Biol. Chem. 251:1009-1014 (1976); Brown, J. L., et al., J. Biol. Chem. 254:1447-1449 (1979)). In lower eukaryotic organisms, about 50% of the soluble proteins are acetylated (Brown, J. L., Int'l. Conor. Biochem. Abstr. (International Union of Biochemistry, Canada) Vol. 11:90 (1979)). These data demonstrate that the N.sup..alpha. -acetyl group is a very important blocking group. It has been suggested that the biological function of this blocking group may be to protect against premature protein catabolism (Jornvall, H., J. Theor. Biol 55:1-12 (1975)) and protein proteolytic degradation (Rubenstein, P. and Deuchler, J., J. Biol. Chem. 254:11142 (1979)). However, in mouse L-cells such N.sup..alpha. -acetylation does not apparently have this biological function (Brown, J. L., J. Biol. Chem. 254:1447 (1979)).
Although a clear general function for N.sup..alpha. -acetylation has not been assessed with certainty, some specific effects for a small number of proteins have been observed. Nonacetylated NADP-specific glutamate dehydrogenase in a mutant of Neurosopra crassa is heat-unstable, in contrast to the acetylated form (Siddig et al., J. Mol. Biol. 137:125 (1980)). A mutant of Escherichia coli, in which ribosomal protein S5 is not acetylated, exhibits thermosensitivity (Cumberlidge, A. G. and Isono, K., J. Mol. Biol. 131:169 (1979)). N.sup..alpha. -acetylation of two of the products from the precursor protein proopiomelanocortin has a profound regulatory effect on the biological activity of these polypeptides; the opioid activity of .beta.-endorphin is completely suppressed, while the melanotropic effect of .alpha.-MSH is increased if N.sup..alpha. -acetylated (Smyth et al., Nature 279:252 (1970); Smyth, D. G. and Zakarian, S., Nature 288:613 (1980); and Ramachandran, J. and Li, C. H., Adv. Enzymol. 29:391 (1967)). Both acetylated and nonacetylated cytoplasmic actin from cultured Drosophila cells participate in the assembly of microfilaments, the latter, however, with less efficiency (Berger et al., Biochem. Genet. 19:321 (1981)). More recently, the rate of protein turnover mediated by the ubiquitin-dependent degradation system was shown to depend on the presence of a free .alpha.-NH2 group at the N-terminus of a protein (Hershko et al., Proc. Nat'l Acad. Sci. U.S.A. 81:9021-9025 (1984) and Bachmair et al., Science 234:179-186 (1986)), suggesting that N.sup..alpha. -acetylation may have a role in impeding protein turnover.
N.sup..alpha. -acetylation is mediated by at least one N.sup..alpha. -acetyltransferase, which catalyzes the transfer of an acetyl group from acetyl coenzyme A to the .alpha.-NH.sub.2 group of proteins and peptides. N.sup..alpha. -acetyltransferases have previously been demonstrated in E. coli (Brot, N., et al., Arch. Biochem. Biophys. 155:475-477 (1973)), rat liver (Pestana, A., et al., Biochemistry 14:1397-1403 (1975); Pestana, A., et al., Biochemistry 14:1404-1412 (1975); Yamada, R., et al., 1st Symposium of the Protein Society 625:34 (1987)), rat brain (O'Donohue, T. L., J. Biol. Chem. 258:2163-2167 (1983)), rat pituitary (Woodford, T. A., et al., J. Biol. Chem. 254:4993-4999 (1979); Pease, K. A., et al., Arch Biochem. Biophys. 212:177-185 (1981); Gembotski, C. C., J. Biol. Chem. 257:10501-10509 (1982); Chappell, M. C., et al., J. Biol. Chem. 261:1088-1091 (1986)), bovine pituitary (Gembotski, C. C., J. Biol. Chem. 257:10501-10509 (1982)), bovine lens (Granger, M., et al., Proc. Natl. Acad. Sci. USA 73:3010-314 (1976)), hen oviduct (Tsunasawa, S., et al., J. Biochem. 87:645-650 (1980)), and wheat germ (Kido, H., et al., Arch Biochem. Biophys. 208:95-100 (1981)). N.sup..alpha. -acetyltransferase enzymes from these sources have, however, never been purified more than 40-fold.